High throughput assays, for identifying candidate compounds which inhibit one or more target proteins in an organism, are most efficient when they include an easily identifiable signal for identifying compounds with the desired inhibitory activity. One such well known system utilizes a responsive promoter, which is activated by the inactivation of the target by a candidate compound, and which is fused to an easily identifiable reporter. When the target protein is inactivated by a candidate compound, the reporter is expressed and the corresponding compound is identified as inhibitory. Such responsive promoters can be identified simply by identifying promoters which are activated by the genetic activation or inactivation of a target gene of interest (e.g., by construction of a temperature-sensitive mutation in the gene).
Responsive promoter screening assays have been used to identify anti-fungal compounds such as azoles which inhibit the egosterol biosynthesis pathway. U.S. Pat. No. 5,527,687; Dixon et al., J. Steroid Biochem. Mol. Biol. 1997 June;62(2-3):165-71. In addition, screens have been developed to screen for inhibitors of protein secretion, membrane stability, protein synthesis and cell wall integrity. Bianchi et al., Appl Environ. Microbiol. 1999 November;65(11):5023-7; Alksne et al., Antimicrob. Agents Chemother. 2000 June;44(6): 1418-27; Shapiro et al., Antimicrob. Agents Chemother. 2002 August;46(8):2490-7; Sun et al., J. Antibiot. (Tokyo). 2002; 55(3): 279-287. These screens are convenient for high-throughput assay systems requiring short incubation times, sub-lethal compound concentrations and the selective induction of the reporter fusion indicates that the compound is perturbing the pathway of interest. Fischer et al., Genome Res. 2004 January;14(1):90-8.
Although some responsive promoters are known and they have been used to identify anti-microbial substances, there still exists a need in the art for the identification of new responsive promoters which may be used to identify other substances of clinical interest.